DESCRIPTION

SETDB1, also named as ESET, KIAA0067 and KMT1E, belongs to the histone-lysine methyltransferase family. It is a SET domain protein with histone H3-K9-specific methyltransferase activity. H3 'Lys-9' trimethylation is coordinated with DNA methylation and represents a specific tag for epigenetic transcriptional repression by recruiting HP1 (CBX1, CBX3 and/or CBX5) proteins to methylated histones. SETDB1 mainly functions in euchromatin regions, thereby playing a central role in the silencing of euchromatic genes. It probably forms a complex with MBD1 and ATF7IP that represses transcription and couples DNA methylation and histone 'Lys-9' trimethylation. Its activity is dependent on MBD1 and is heritably maintained through DNA replication by being recruited by CAF-1. SETDB1 regulates histone methylation, gene silencing, and transcriptional repression. It has been identified as a target for treatment in Huntington Disease, given that gene silencing and transcription dysfunction likely play a role in the disease pathogenesis.

PRINCIPLE OF THE ASSAY

Sandwich ELISA structure (Detection antibody labeled with HRP) A capture antibody is pre-coated onto the bottom of wells which binds to analyte of interest. A detection antibody labeled with HRP also binds to the analyte. TMB acts as the HRP substrate and the solution color will change from colorless to blue. A stop solution containing sulfuric acid turns solution yellow. The color intensity is proportional to the quantity of bound protein which is measurable at 450 nm with the correction wavelength set at 630 nm.

PRECISION

Intra-assay Precision (Precision within an assay) Three samples of known concentration were tested 8 times on one plate to assess intra-assay precision. Inter-assay Precision (Precision between assays) Three samples of known concentration were tested in 16 separate assays to assess inter-assay precision.

Intra-assay Precision

Sample	n	Mean (ng/mL)	SD	CV%
1	8	4.66	0.18	3.77
2	8	1.00	0.07	7.30
3	8	0.59	0.03	4.82

Inter-assay Precision

Sample	n	Mean (ng/mL)	SD	CV%
1	16	4.64	0.18	3.82
2	16	1.01	0.06	5.57
3	16	0.61	0.04	7.24

RECOVERY

The recovery of human SETDB1 spiked to three different levels throughout the range of the assay in cell lysate was evaluated.

Sample Type		Average% of Expected	Range (%)
Cell lysate	1:64	108	106-110

LINEARITY

To assess the linearity of the assay, samples were diluted with the appropriate Sample Diluent to produce samples with values within the dynamic range of the assay. (The cell lysate was initially diluted 1:4.)

		Cell lysate
1:2	Average% of Expected	100
	Range (%)	-
1:4	Average% of Expected	112
	Range (%)	110-113
1:8	Average% of Expected	99
	Range (%)	97-101
1:16	Average% of Expected	130
	Range (%)	129-131

CALIBRATION

This immunoassay is calibrated against a highly purified recombinant human Human SETDB1 Sandwich ELISA Kit produced at Sunlong Medical™.